1. Field of Invention
This invention relates to a method for identifying inhibitors of botulinum neurotoxins.
2. Background of the Invention
Botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise two polypeptide chains connected via a disulfide linkage. The light chain (˜50 kDa) is disulfide linked to a heavy chain (˜100 kDa). The anaerobic bacterium Clostridium botulinum produces seven immunologically distinct but structurally similar neurotoxins designated BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F and BoNT/G (collectively, “BoNTs”). After synthesis, highly active neurotoxin is generated by proteolytic cleavage of the clostridial neurotoxins.
These neurotoxins inhibit neurotransmitter release at distinct synapses, which causes two severe neuroparalytic diseases, tetanus and botulism. Many aspects of the cellular and molecular modes of action of these toxins have been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Internalization of toxin is a rapid event and the toxin shows persistent catalytic activity within neurons. Subsequently, the light chain of the neurotoxin is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: (1) synaptosomal associated protein of 25 kDa (SNAP-25); (2) synaptobrevin, also called vesicle associated membrane protein (VAMP); and (3) syntaxin. Specifically, BoNT/A, BoNT/E and BoNT/C cleave SNAP-25; BoNT/C also cleaves syntaxin. BoNT/B, BoNT/D, BoNT/F, BoNT/G cleave synaptobrevin/VAMP. Tetanus neurotoxin cleaves synaptobrevin/VAMP, at the same cleavage site as BoNT/B. See, Schmidt J J, et al., supra; Anne C, et al., Anal Biochem (2001 291:253-61).
The location of the enzymatic subunit of the clostridial neurotoxins has been mapped to the light chain, which has Zn endopeptidase activity. The binding and translocation motifs in a BoNT are located within the heavy (H) chain. All of the BoNT serotypes bind to receptors/acceptors on the presynaptic terminals of motor neurons at the neuromuscular junction. Schiavo G, et al., (1993) FEBS Lett 335:99-103. The binding of the BoNT to the presynaptic terminal is mediated by the C-terminal domain of the heavy chain (HC) of the toxin. Schiavo G, et al., J Biol Chem (1993) 268: 23784-7 and Schiavo G, et al., Nature (1992) 359: 832-5. Binding is followed by endocytosis of the toxin into vesicles at the presynaptic terminal. As the endocytotic vesicle is acidified, the N-terminus of the HC forms a pore in the vesicle membrane. The light chain (LC) disassociates from HC to act as a zinc-dependent protease that cleaves and inactivates SNARE proteins essential for exocytosis of neurotransmitter. Arnon S S, et al., JAMA 2001, 285:1059-70. In the case of BoNT/A (the most potent and persistent of the BoNTs) the substrate is SNAP-25, a SNARE protein which resides on the cytoplasmic surface of the presynaptic membrane. See, Foran P, et al., Biochemistry (1996) 35:2630-6; Lewis J, et al., Nat Med (1999) 5:832-5; and Schmidt J J, et al., Anal Biochem (2001) 296:130-7.
The botulinum neurotoxin cleaves the substrate proteins at highly specific sites. BoNT/A cleaves SNAP-25 at residues 197/198 (amino acids QR). See, Foran P, et al., Biochemistry (1996) 35:2630-6; and Lewis J, et al., (1999) supra. BoNT/E cleaves SNAP-25 at residues 180/181 (amino acids RI).
The unique specificities of BoNT/A and BoNT/E for SNAP-25 was suggested to be directed through the recognition of a nine residue sequence, termed the SNARE motif. The SNARE motif is about 50 amino acids in length and assumes a coiled confirmation. The SNARE motif in SNAP-25 is common to the other two SNARE proteins: VAMP and syntaxin. SNAP-25, VAMP and syntaxin are the only known substrates of the seven clostridial neurotoxins. There are four copies of the SNARE motif present in SNAP-25. Studies on the interaction of SNAP-25 with BoNT/A and BoNT/E showed that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognize SNAP-25. Washbourne P et al., FEBS Lett. (1997) 418:1-5. The full kinetic activity of BoNT/A and BoNT/E for SNAP-25 requires at least one SNARE motif. Although the copy of the SNARE motif that is proximal to the SNAP-25 cleavage site is clearly involved in recognition with BoNT/A and BoNT/E, in its absence, other more distant copies of the motif are able to support proteolysis. Id.
The proteolytic attack at specific sites in the protein targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics. These alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release caused by the neurotoxins.
The botulinum neurotoxins (BoNTs) are some of the most potent and persistent toxins known and can be delivered by an oral or inhalation route. These properties have contributed to attempts by others to use BoNT as a bioweapon. No effective antidote for BoNT intoxication is available. Current therapy consists primarily of long term ventilator support, although early administration of hyperimmune antiserum within the first 12 hours can shorten the duration of paralysis. This therapy currently involves administration of horse serum derived antibodies with the risks of anaphylactic reaction. Human hyperimmune antiserum is used to treat infantile botulism. Human hyperimmune antiserum is too limited a source for use in a bioterrorism attack involving BoNT. Monoclonal IgG antitoxins are being pursued for BoNT therapy, but at least three different monoclonal antibodies are required to inhibit each of the serotypes of botulinum neurotoxin. The cost of producing an oligoclonal treatment consisting of 15-18 monoclonal antibodies is not commercially feasible.
Immunization is currently the major biodefense strategy against BoNT attacks. Although vaccination can clearly protect against the paralytic effects of the toxin, there are clear limitations to this strategy which include: 1) the need to vaccinate a large at risk population to prevent disease in even a small number of exposed individuals; 2) active vaccination must be accomplished well before exposure to the toxin; 3) strains of BoNT can be engineered for bioterrorism, that can evade immune defense or delivered by viral vector overcoming host immunity (See Fishman P S, et al., Nat Toxins 1999, 7:151-6), and; 4) vaccination will interfere with the potential future use of BoNT for medical conditions and deny the current standard of care to immunized patients. Oyler G A, et al., IBRCC (2001).
An alternative strategy to vaccination against BoNT is the development of a clinically useful antidote. Oyler G A, et al., Interagency Botulinum Research Coordinating Committee, 2001. This strategy opens a wide array of possibilities based on the understanding of the molecular pathogenesis of intoxication.
Methods to detect botulinum neurotoxin's catalytic activity have been based on detecting SNARE protein cleavage products in vitro. See, for example, Schmidt U.S. Pat. No. 5,965,699, the contents of which are hereby incorporated by reference in their entirety.
The blocking proteolytic activity of the catalytic light chain is a candidate for treatments to inhibit and terminate the action of the toxin. SNARE protein cleavage is a late event in intoxication.
Rapid replenishment of SNARE proteins normally occurs and could result in early restoration of neuromuscular synaptic function. Inhibitors that are able to reach the site of action in the cytosolic compartment of the pre-synaptic terminal of the neuromuscular junction (unprotected by the blood-brain/nerve-barrier) could decrease the neurotoxin's effect in infected individuals. There is a need for a method to identify a clinically relevant botulinum catalytic inhibitor that penetrates to the intracellular site of action of the toxin and is non-toxic to living cells. Therefore, a need exists for a method for screening inhibitors of botulinum neurotoxin type A (BoNT/A), to identify neurotoxin inhibitors that function both in vitro and in living cells. There is also a need for a method of screening inhibitors of botulinum neurotoxin type E (BoNT/E), type C (BoNT/C), type B (BoNT/B), type D (BoNT/D), type F (BoNT/F) and type G (BoNT/G) that can be used to identify neurotoxin inhibitors that function both in vitro and in living cells. In order to facilitate the identification and development of such botulinum toxin inhibitors, there is a need for a system to rapidly assess botulinum toxin catalytic activity.